Mass spectrometry (MS) is a central analytical technology that finds a large variety of applications in a broad range of fields, especially when coupled with a separation technique such as gas chromatography (GC) or liquid chromatography (LC). GC-MS has become the most widely used form of mass spectrometry.
GC-MS is generally characterized by very good sensitivity, excellent separation resolution, very good molecular identification capability through the rich ion fragmentation pattern and extensive libraries available for identification, relative ease of use and low cost. However, GC-MS suffers from a major limitation: its inability to analyze thermally labile (and relatively non-volatile) compounds that tend to dissociate in the GC injector, column or MS electron impact ion source. This drawback is especially severe when relatively large bio-molecules and drugs are encountered.
Thus, various methods of LC-MS were developed for the analysis of these compounds, and LC-MS is now experiencing rapid development and growth in its use and applications. The major problem in the coupling of an LC to the MS is in the need to avoid large solvent load on the MS high vacuum system, coupled with the need to preserve molecular integrity. Accordingly, the type of LC-MS is usually named after its MS interface and ionization technique. Today, the major LC-MS techniques are:
1. Particle Beam LC-MS (PB-LC-MS)
In this method, the LC effluent is sprayed through a thermally assisted or pneumatic nebulyzer into a drying desolvation chamber to form small droplets or particles that expand through a supersonic nozzle into a differentially pumped vacuum chamber before entering the MS ion source. The heavy sample particles (hence the name particle beam) formed after solvent vaporization move preferentially forward, while the solvent molecules are pumped away. Sample/solvent separation factor of over 105 can be achieved. The sample particles are thermally vaporized inside the electron ionization (EI) ion source, and the sample compounds are ionized as gas phase molecules in thermal equilibrium with the heated ion source walls. The EI ion source is a standard ion source with an added heated plate to assist with the intra ion source thermal vaporization of the particles. PB-LC-MS is a useful technique especially due to its EI mass spectra, which provide library-searchable EI mass spectra for easy molecular identification. However, PB-LC-MS is limited in its ability to analyze very thermally labile compounds, due to the intra-ion source thermal vaporization stage. Furthermore, compounds with low volatility tend to exhibit ion source-related peak tailing, due to lengthy intra-ion source adsorption-desorption cycles. This peak tailing can be reduced by further heating the ion source, but with the major penalty of excessive molecular and/or molecular ion dissociation. PB-LC-MS also suffers from non linear signal dependence on the sample concentration (and matrix) due to variation in the particle transmission versus its size due to partial vaporization of small sample compounds that are pumped away and lost with the solvent.
2. Atmospheric Pressure Chemical Ionization (APCI)
In APCI, the LC effluent is sprayed and ionized in a zone of corona discharge, at about 1 atmosphere. The solvent molecules and other gases are ionized and then the vaporized sample compounds are ionized through a series of atmospheric pressure charge transfer and chemical ionization processes. The sample ionization efficiency is very high, but typically only 10−4 of the ions are transferred to the MS through a 100μ nozzle or ion transfer tube. In contrast to the particle beam method, APCI involves with high-pressure sample vaporization. APCI is a soft ionization technique that finds growing use. However, the existence of mostly M+ ions is a limitation that is usually overcome by the use of costly and complex MS-MS instrumentation that enables molecular ion dissociation and provides fragment information. In addition, APCI is relatively ineffective for the ionization of non-polar compounds; its ionization efficiency is compound-dependent and therefore non-quantitative, and thus requires compound-specific calibration for quantification.
3. Electrospray LC-MS (ES-LC-MS)
ES-LC-MS has recently become the most popular LC-MS method. It is based on spray formation from a highly charged needle and spontaneous ion evaporation from the highly charged droplets. The main attribute of electrospray (ES) is the possible formation of multi-charged molecular ions that enable very high mass determination up to about 105 Dalton. It is also currently the most sensitive LC-MS method. ES can also be used with small molecules, but it suffers from a non-uniform response (non-quantitative detection) that may vary substantially among different compounds and has to be optimized for each molecule separately. In general, ES sensitivity is reduced for both small molecules and non-polar compounds. MS-MS instrumentation is also desired with ES, in order to provide fragment information and to enable better identification capability in view of the lack of ES mass spectral libraries. Adduct ion formation and complex matrix effects also hamper the effectiveness of both APCI and ES.
The use of supersonic molecular beams (SMB) for sampling and ionization in mass spectrometry was explored, aimed at improving all aspects of GC-MS with special emphasis on the development of improved, fast GC-MS. Supersonic molecular beams are characterized by the following features, which are of importance to mass spectrometry:
Extreme Intra-molecular Vibrational-rotational Supercooling:
Upon the expansion of organic compounds from a supersonic nozzle into a vacuum system, significant vibrational and rotational supercooling occurs. Thus, upon collimation a supersonic molecular beam is formed with vibrationally cold sample molecules for its further ionization by electrons or on a surface.
This intra-molecular cooling considerably improves the level of mass spectral information provided by electron ionization, when the sample compounds are ionized as vibrationally cold molecules contained in the supersonic molecular beam. The molecular ion abundance is largely enhanced and it is practically always exhibited, combined with the library-searchable fragment ions. Peak tailing due to lengthy intra ion source adsorption-desorption cycles is eliminated and matrix interference is reduced at the molecular and high mass fragment ions. Isomer and other structural effects are amplified and isotope abundance and elemental information is enabled. This enhanced information is provided even for thermally labile and relatively non-volatile compounds.
Unidirectional Motion with Controlled Hyperthermal Kinetic Energy up to 30 eV:
This directional kinetic energy enables a very effective ionization method called Hyperthermal Surface Ionization (HSI). HSI is based on the hyperthermal surface scattering of the sample compound from a suitable surface such as rhenium oxide, having a high surface work function. In HSI, the molecular kinetic energy is used to effectively bridge upon the surface ionization potential (IP-φ). Thus, the hyperthermal surface scattering is followed by spontaneous molecular ionization that can be very effective for compounds with relatively low molecular ionization potentials, such as polycyclic aromatic hydrocarbons (PAHs) or drugs. HSI is also a selective ionization method that is thus effective for the detection of drugs and PAHs in complex matrices, due to the reduced efficiency of aliphatic compounds ionization. HSI is potentially the most efficient mass spectrometric ionization due to very high ionization yield (up to 10%), its unique fragmentation pattern which can exhibit a single molecular or fragmented ion and the reduced vacuum background of the thermal molecules.
High Flow Rate (100-500 ml/min) Atmospheric Pressure Sample Inlet Capability, Combined with Heavy Species Focusing in the Beam Axis (Jet Separation):
This feature simplifies the transfer of the sample compounds from a GC or LC, according to the present invention.
HSI is potentially the most efficient mass spectrometric ionization method, due to its very high ionization yield (up to 10%), its unique fragmentation pattern which can exhibit a single molecular or fragmented ion, and the reduced vacuum background of the thermal molecules.
Up to now, the technique of mass spectrometry with supersonic molecular beams (SMB-MS) was successfully employed with gas phase samples, provided either from a direct sample introduction device after thermal vaporization, or from a gas chromatograph. However, its coupling with liquid samples of thermally labile compounds or with the output of an LC was not performed, despite the considerable merits of LC-MS, due to several major problems that must be considered and overcome:    a) The problem of intact vaporization of thermally labile compounds is central to the achievement of this goal. This is a major and complex problem that cannot be overcome by any standard, known approach.    b) Isolated vaporized sample compounds must be vibrationally cooled, avoid shock wave heating, collimated, properly enriched in the SMB, and be ionized while contained in the SMB for the preservation of the merits of SMB.    c) The liquid solvent load on the vacuum pumps needs to be considered, including its effect on the molecular cooling, aerodynamic acceleration and jet separation efficiency.    d) Peak tailing from all sources must be eliminated, combined with molecular cooling in the supersonic beam.    e) Cluster and adduct ion formation must be avoided or minimized.    f) All of the above items must be achieved with high sample transfer and ionization efficiency.